Affinity column and process for detection of low molecular weight toxic substances

ABSTRACT

An affinity matrix and a method for the detection of low molecular weight compositions such as aflatoxins are provided utilizing specific monoclonal IgM antibody having an affinity constant not less than about 1×10 9  liters per mole. Methods for the preparation and use of such affinity matrices are also given. The detection is rapid, accurate, reproducible, and allows for quantitative recovery of the composition of interest.

RESEARCH SUPPORT

The investigations reported herein were supported by a grant from theNational Institutes of Health and American Cancer Society, MassachusettsDivision.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of co-pendingapplication Nos. 706,983 and 706,984, both filed on Feb. 28, 1985.

FIELD OF THE INVENTION

The invention is concerned with non-invasive immunological screeningprocedures for assessing the exposure of humans and animals toenvironmentally occuring toxins and is particularly directed toimmunological compositions and processes for the detection of mutagensand carcinogens frequently encountered in many areas of the world.

BACKGROUND OF THE INVENTION

The incidence and effect of exposure to toxic substances by humans andother animals via food, water, and air is of critical importance to oursurvival. Within the general class of toxic substances, the detection ofsmall molecular weight (1,000 daltons or less) mutagens and/orcarcinogens such as aflatoxins, fluoranthene, nitropyrene,nitrofluoranthene, nitrochrysene, and aminobiphenyls have becomeespecially important. In particular, non-invasive screening proceduresfor assessing the exposure of humans to substances such as aflatoxinsrequire the ability to quantify both the toxin and its metabolites,especially covalent adducts formed with DNA and proteins, in body fluidssuch as serum and urine.

Aflatoxins are a typical example of the toxic and carcinogenic compoundswithin this class. Aflatoxins are secondary fungal metabolites,mycotoxins, which are produced by Aspergillus flavus and Aspergillusparasiticus and are structurally a group of substituted coumarinscontaining a fused dihydrofurofuran moiety. Aflatoxins occur naturallyin peanuts, peanut meal, cottonseed meal, corn, dried chili peppers andthe like; however the growth of the mold itself does not predict thepresence or levels of the toxin because the yield of aflatoxin dependson growth conditions as well as the genetic requirements of the species.A variety of aflatoxins--types B₁, B₂, G₁, G₂, M₁ and M₂ --have beenisolated and characterized. Aflatoxin B₁ (hereinafter "AFB₁ ") is themost biologically potent of these compounds and has been shown to betoxic, mutagenic and carcinogenic in many animal species. This mycotoxinis a frequent contaminant of the human food supply in many areas of theworld and is statistically associated with increased incidence of humanliver cancer in Asia and Africa in particular [Busby et al., inFood-Born Infections and Intoxications (Riemann and Bryan, Editors)Second Edition, Academic Press, Inc., 1979, pp. 519-610; Wogan, G. N.Methods Cancer Res. 7:309-344 (1973)].

AFB₁ also forms covalently linked adducts with guanine in DNA afteroxidative metabolism to a highly reactive 2,3-exo-epoxide, the majoradduct product being 2,3-dihydro-2-(N⁷ -guanyl)-3-hydroxy-aflatoxin B₁(hereinafter identified as "AFB₁ -N⁷ -Gua") [Lin et al., Cancer Res.37:4430-4438 (1977); Essigman et al., Proc. Natl. Acad. Sci. U.S.A.74:1870-1874 (1977); Martin et al., Nature (London) 267:863-865 (1977)].The AFB₁ -N⁷ -Gua adduct and its putative derivatives [2,3-dihydro-2-(N⁵-formyl-2', 5', 6'-triamino-4'-oxo' N⁵ -pyrimidyl)-3-hydroxy-aflatoxinB₁ ] (hereinafter "AF-N⁷ -Gua") have been identified in a wide varietyof tissues and systems such as rat liver in vivo, cultured humanbronchus and colon, and human lung cells in culture after acute orchronic administration [Haugen et al., Proc. Natl. Acad. Sci. U.S.A.78:4124-4127 (1981)].

Some investigations regarding quantitation of aflatoxin B₁ and itsmetabolites including its DNA adduct have been conducted usingimmunological techniques and monoclonal antibodies [Hertzog et al.,Carcinogensis 3:825-828 (1982); Groopman et al., Cancer Res.42:3120-3124 (1982); Haugen et al., Proc. Natl. Acad. Sci. U.S.A.78:4124-4127 (1981)]. Similar research has been conducted utilizingimmunological techniques and reagents for other low molecular weighttoxins found in our environment [Johnson et al., J. Analyt. Toxicol.4:86-90 (1980); Sizaret et al., J.N.C.I. 69:1375-1381 (1982); Hu et al.,J. Food Prot. 47:126-127 (1984); and Chu, J. Food Prot. 47:562-569(1984)]. Nevertheless, insofar as is presently known, the development ofa general non-invasive screening procedure for assessing the exposure ofhumans and animals to such environmentally occurring carcinogens has notbeen achieved.

SUMMARY OF THE INVENTION

The present invention comprises two distinct parts, each of whichrepresents a major advance and contribution to the invention as a whole.One part of the invention is an affinity matrix material for thedetection of a toxic substance such as aflatoxin B₁ in a test samplecomprising a solid phase sorbent material and high affinity IgM antibodyspecific for the toxic substance, the IgM antibody being bound to thesorbent material. This aspect of the invention provides a novel andwidely useful method for purifying samples for testing of toxins by useof a specific monoclonal antibody bound to a sorbent material.

In addition, the invention comprises a method for detecting a lowmolecular weight substance such as aflatoxin B₁ in a fluid samplecomprising the steps of preparing an affinity matrix comprised of ahomogenous, high affinity IgM antibody specific for the toxic substanceof interest, the antibody being immobilized onto a solid phase sorbentmaterial; combining the sample with the affinity matrix such that thesubstance in the sample is retained by the IgM antibodies; adding areleasing agent to the affinity matrix; and detecting the presence ofthe toxic substance in the effluent.

DETAILED DESCRIPTION OF THE DRAWING

The present invention may be more fully and easily understood when takenin conjunction with the accompanying drawing, in which:

FIG. 1 is a graph illustrating a competitive radioimmunoassay using ³H-AFB₁ tracer and a variety of aflatoxins to measure the specificity ofIgM antibody;

FIG. 2 is a graph illustrating a competitive radioimmunoassay using ³H-AFB₁ tracer in combination with aflatoxin B₁ and the major metabolicaflatoxin-DNA adducts using the IgM antibody;

FIG. 3 is a schematic flow outline for the detection and isolation oftoxins in general; and

FIG. 4 is a schematic flow outline for an apparatus for automaticallyconducting an aflatoxin assay procedure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is useful for the detection and isolation of toxicsubstances generally when they present two critical characteristics:first, the toxic substance has a molecular weight not greater than about1000 daltons; second, that the toxic substance, either alone or incombination with other compounds, is able to induce an immunologicalresponse in vivo when introduced into an animal subject.

The first requirement, a molecular weight not greater than about 1000daltons, is easily determinable for any toxic substance. The term "toxicsubstance" as used herein includes all compounds which have been shownto be harmful, hazardous, or destructive, either by in vitro tests or invivo determinations (including epidemological studies). Most notable arethose compounds shown to be mutagenic, genotoxic, and/or carcinogenic asthese terms are used and recognized in this art.

The second characteristic, the ability of the toxic substance to inducean immunological response after introduction into a test animal in vivo,relies on its ability primarily (but not exclusively) to induce antibodyformation. It is recognized that often the production of antibodieseither precedes or is subsequent to other immunological responses andthat the ability to induce antibody production is dependent upon methodsfor the preparation and introduction of the antigen (or hapten) whichdirectly affect the quantitative and qualitative antibody response. Forthis reason, so long as some type of immunological response is observed,it is understood and accepted that production of specific antibodies canbe achieved using the techniques and manipulations presently known inthis art.

The invention therefore applies generally to all toxic substances whichmeet the two requisite criteria. Specifically included within this classof compounds are aflatoxins (regardless of designation) and also thosepolynuclear aromatic hydrocarbons exemplified by fluoranthene,nitropyrene, nitrosopyrene, nitrofluoranthene, nitrochrysene, andaminobiphenyls (and their derivatives). This general category ofaromatic hydrocarbons and each of the specific examples identified arerecognized immunogens and are mutagenic/carcinogenic in nature. Theinvention is particularly useful for studies of aflatoxin B₁ andaflatoxin M₁. An entire listing of toxic substances which are alsoimmunogens is extensive, varied in chemical composition and structure,and expanding in view of the ever increasing list compiled by the U.S.Dept. of Health and Human Services. In view of this, a true and completelisting is neither possible nor desirable and will not be attemptedhere. Instead a representative example, aflatoxin B₁, will be the toxicsubstance of choice which is described for use and detection by thepresent invention. It is expressly understood, however, that the presentinvention is not limited to this working example nor to those othertoxic substances specifically identified above but rather is suitableinstead generally for the entire class of toxic substances which meetthe two essential requirements.

In view of aflatoxin being the descriptive example herein for all toxicsubstances of interest as a class and to promote clarity and a completeunderstanding of the invention in all its component parts, the detaileddescription will describe each part of the invention independently inseparate sections. It will be recognized also that substantial portionsof the detailed description relating to the preparation of hybridomasand the production of high affinity IgM antibodies are identical to andshared in common with culture deposit ATCC No. HB8719 and the copendingpatent application of Groopman, et al., Ser. No. 706,983 filed on Feb.28, 1985.

I. Preparation of Aflatoxin Immunogen

The preferred immunogen is a composition in which aflatoxin B₁ has beenconjugated to bovine gamma globulin (hereinafter "BGG"). Initially, BGG(commercial grade) was dissolved in phosphate buffered saline(hereinafter "PBS"), pH 7.0 at a concentration of 10 milligrams permilliliter (hereinafter "mg/ml"). The aflatoxin B₁ (hereinafter "AFB₁ ")was conjugated to BGG using a modification of the procedure employingM-chloroperoxybenzoic acid [Martin et al., Nature London 267:863-865(1977)]. Typically one milligram (hereinafter "mg") of AFB₁ (3.2 μmoles)was dissolved in 2.0 ml of methylene chloride and added to a 5 molarexcess of M-chloroperoxybenzoic acid (mCPBA) which had been previouslydissolved in 2.0 ml of methylene chloride. The BGG in PBS buffersolution was added to this reaction mixture to produce a 5 molar excessof aflatoxin relative to the protein content. The reaction mixture wasvigorously stirred overnight at ambient temperature and the reactionterminated subsequently by centrifugation at 2,000 times gravity for 20minutes. The aqueous ephiphase (supernatant) containing the modified BGGprotein was then extensively dialyzed against PBS at pH 7.4. The levelof modification of the globulin protein was quantified by measuring theabsorbance at 362 nanometers, using a molar extinction coefficient of18,000. The reaction product of such a preparation AFB₁ -BGG,demonstrated an average of 40-50 AFB₁ residues to be bound per moleculeper BGG.

It should be noted that AFB₁ may be conjugated to other carriers to formthe immunogen using the procedure described above. For example, thepreparation of AFB₁ coupled to bovine serum albumin (hereinafter "BSA")is a useful alternative; the average level of binding the AFB₁ to bovineserum albumin, however, is usually only 20-30 molecules of alfatoxin permolecule of BSA. Other useful carriers may be combined with AFB₁ to forma conjugate using reaction methodologies presently known in the art.Exemplifying such conjugates are the following: AFB₁ - BGG (mCPBA);single strand AFB₁ - DNA - meBSA; AFB₁ -N⁷ -Gua-BGG-PABA (para aminobenzoic acid); AFB₁ -carboxymethyloxime-KLH (Kehole Limpet Hemocyanin);and AFB₁ -poly Gua-BGG.

II. Immunization of Mice

Female BALB/By CJ (Jackson Labs), approximately 16 weeks of age, wereimmunized with AFB₁ -BGG immunogen which had been dissolved in PBS andemulsified with an equal volume of complete Freund's adjuvant. Using twogroups of five mice each, immunization was performed by intraperitonealinjection of 37.5 μg of AFB₁ -BGG (with adjuvant) or 12 μg AFB₁ -BGG(with adjuvant) in a final volume of 0.2 ml PBS. At 5 weeks and 9 weekspost initial injection, each mouse received an identical quantity ofAFB₁ -BGG emulsified with incomplete Freund's adjuvant. Approximately 10days after the second injection, serum samples were taken from eachmouse via tail bleeding and were assayed for anti-aflatoxin antibodyactivity by ELISA immunoassay determination. For those mice showing thepresence of specific antibody in their serum, each was given a finalimmunization of the identical AFB₁ -BGG again in 0.1 ml of PBS injectedinto the tail vein 3 days prior to sacrifice of the animal.

The ELISA immunoassay alo was used to determine the presence of specificantibodies against AFB₁ in mouse sera (and subsequently to identifyspecific hybridomas); these assays were modifications of methodspreviously described in the art [Haugen et al., Proc. Natl. Acad. Sci.U.S.A. 78:4124-4127 (1981); Groopman et al., Cancer Res. 42:3120-3124(1982)]. Briefly summarizing the procedure, AFB₁ -BSA was dissolved inPBS at a concentration of 2.0 μg/ml and 50 μl of this fluid mixture wasadded to each well of a polyvinyl microtiter plate and allowed toincubate for 2-4 hours at ambient temperature. Other wells in themicrotiter plate received 50 μl of BSA in PBS at a concentration of 2μg/ml and served as controls. The fluid in each well was then aspiratedand each well washed 3 times with tap water. Subsequently each wellreceived a PBS solution containing either 0.2% BSA or 0.2% gelatin (typeIV, Sigma) and the plates were allowed to incubate for an additionalhour at ambient temperature. This procedure was designed to limitnon-specific binding of antibodies. The plates were then washed in tapwater and 50 μl aliquots mouse serum samples (or hybridoma medium) addedto each well. To titer the mouse sera, dilutions in PBS containing 10%fetal calf serum were prepared over a range from 1:50-3:50,000 incontinuing three-fold dilutions. When using hybridoma media, 50 μlaliquots were used without dilution. In either case, the microtiterplates were then incubated for 90 minutes at 37° C., after which theywere thoroughly washed with tap water. Specific antibodies that becamebound to the surface of each well were detected by adding 50 μl of a1:200 dilution of rat anti-mouse kappa antibody coupled to alkalinephosphatase to each well followed by incubation of 4 hours at roomtemperature or incubation overnight at 4° C. The wells in each platewere then rewashed with tap water. 100 μl per well of 1.0 mg/mlp-nitrophenyl phosphate solution (Sigma) prepared in 0.1M diethanolaminebuffer, pH 9.8 then was added and allowed to react for 1-2 hours.Quantitative measurement of the p-nitrophenol reaction product wasperformed by measuring the absorbance of the assay well at 405nanometers using a microtiter plate reader (Dynatech Labs).

The isotypes of the monoclonal antibodies (that is the determination andidentification of different antibody heavy chain class) were determinedin a non-competitive ELISA methodology using a commercially purchasedkit for mouse immunoglobulin subtype identification (Boeringer-MannheimCompany).

III. Preparation of Hybridomas and Isolation of Monoclonal AntibodyProducing Cells

The female BALB/By CJ mice previously immunized with AFB₁ -BGG incomplete Freund's adjuvant were tested for production of significantanti-aflatoxin B₁ serum titers using the non-competitive ELISAmethodology as described above. Those mice showing high titers weresacrificed and hybridomas prepared following the procedures previouslydescribed in Marshak-Rothstein et al., J. Immun., 122:2491-2497 (1979).The myeloma cell line used for cell fusion were SP2/0 cells which weremaintained Dulbecco's Modified Eagles medium (hereinafter "DME" medium)supplemented with 20% (volume/volume) fetal calf serum, 580 microgramsper ml (hereinafter "μg/ml") glutamine, 10 units/ml penicillin, 100μg/ml streptomycin, and non-essential amino acids (Gibco). The mice weresacrificed and spleen cell suspensions prepared using Hanks' balancesalt solution buffered with 0.01M phosphate, pH 7.2 (hereinafter"HPBS").

The spleen cells from these mice were fused with SP2/0 myeloma cellsusing a modification of the Gefter et al. procedure [Somatic Cell Genet.3:321 (1977)]. Unless stated otherwise, all centrifugations wereperformed at 700 times gravity for 5 minutes at room temperature.Preferably, 5×10⁶ SP2/0 myeloma cells and 5×10⁷ immune spleen cells werecombined in a round bottom plastic tube, centrifuged, resuspended in 10ml of serum free DME medium and centrifuged again. The supernatant wascarefully discarded and the centrifuge tube tapped sharply to dispersethe residual cell pellet. The cells were then exposed to 0.5 ml of a 30%(volume/volume) solution of polyethylene glycol 1000 (Baker ChemicalCompany) in serum free DME for 6 minutes. During this 6 minute period,the cell suspension was gently centrifuged (150×gravity for 3 minutes).4.0 ml of serum free DME was then added to the cell pellet and the cellsagain resuspended by tapping the tube. The contents of the tube weretransferred to 100×17 mm Petri dishes and cultured in DME mediumcontaining 20% fetal calf serum for 1 day. The cells were thencentrifuged again and resuspended in growth medium containinghypoxanthine, aminopterin and thymidine (hereinafter "HAT medium"). 0.1ml aliquots of the cells were then distributed into the wells of flatbottom microtiter dishes, each aliquot containing approximately 10⁵SP2/0 l cells. After one week's incubation, 0.05 ml of growth mediumcontaining only hypoxanthine and thymidine (hereinafter "HT medium") wasadded to each well. Cultures were screened for specific anti-AFB₁antibody activity two weeks post fusion using the ELISA immunoassaytechnique described earlier.

Hybridomas secreting IgM antibodies of high affinity specific for AFB₁were grown as ascites tumor cells in BALB/C mice which had beenpreviously injected with 0.5 ml pristane (Aldrich). The hybridomasgrowing within the mice produced large quantities of specific IgMantibodies which were harvested and collected as ascites fluid from eachmouse before it died. The collected fluid from these animals was pooledand either used directly in the immunoassays or further purified bysaturated ammonium sulfate precipitation and dialysis against PBS. Grosspathological examination showed that all mice died as a result ofwidespread tumor invasion--that is growth of the injected hybridomacells.

Some of the monoclonal antibodies obtained by this method were shown tobe a high affinity IgM antibodies having specificity for the AFB₁molecule. Values for the affinity constant for this antibody weredetermined to be 8×10⁸ and 1×10⁹ liters per mole by Scatchard plotanalysis and by the method of Muller [J. Immunol. Meth., 34:345-352(1980)]. The values were derived from data obtained following acompetitive RIA procedure as follows:

The assay routinely employed 300 microliter (hereinafter "μl") totalvolume of which 100 μl consisted of [³ H]-aflatoxin B₁ tracer (specificactivity 3.4 Ci/mmol) purchased from Moravek Biochemicals. The tracerconcentrate was diluted in 1% normal mouse serum containing 0.1% BSA inPBS to a level of about 20,200 cpm/100 μl. The monoclonal antibody wasdiluted to a concentration which precipitated 30-50% of the aflatoxin B₁tracer. The antibody was added to the reaction mixture in 100 μlaliquots which contain 10% fetal calf serum in PBS. The test sample,consisting of non-radiolabelled aflatoxin B₁ or its metabolitesincluding the major aflatoxin B₁ -DNA adducts AF-N⁷ -Gua and AF-FAPyr,were added to the tube in 100 μl volumes. The reaction mixture wasincubated at ambient temperature for two hours after which the mixturewas brought to a 1.0 ml volume with PBS. An equal volume of ice coldsaturated ammonium sulfate solution was then added after which thesample was mixed and allowed to stand on ice for 15 minutes. The samplewas then centrifuged for 15 minutes at 2000×gravity and the percent ofinhibition in the reaction determined using the Muller methodology.

The specificity of the IgM antibody for AFB₁ and its metabolites areshown in FIGS. 1 and 2. It should be noted that these results wereobtained using purified IgM antibody of high affinity which wasinitially fractionated by precipitation with saturated ammonium sulfatefollowed by dialysis against PBS. The antibody was then purified by highpressure liquid chromatography in a steric exclusion which tookadvantage of the high molecular weight (900,000 daltons) of the IgMantibody for its separation and isolation. It is the characteristics andproperties of this purified IgM antibody whose specificity and highaffinity constants were then utilized as described herein. Using thecompetitive RIA methodology, The 50% inhibition levels for aflatoxin B₁,aflatoxin B₂ and aflatoxin M₁ were found to be 3.0 picomoles whereasthose for aflatoxin G₁, aflatoxin G₂, and aflatoxin Q₁ were 60.0, 84.0,and 275.0 picomoles respectively. The data shown in FIG. 2 identifiesthe specificity and 50% inhibition point for the IgM antibody foraflatoxin B₁ and the two major aflatoxin-DNA adducts, AF-FAPyr and AF-N⁷-Gua. The 50% inhibition values were 3.0, 24.0, and 89.0 pmolesrespectively. The data therefore indicates that this IgM antibody isapproximately four times more sensitive in detecting the AF-FAPyr adductthan the AF-N⁷ -Gua adduct.

IV. Affinity Matrix Comprising Immobilized Anti-AFB₁ IgM MonoclonalAntibody

An affinity matrix material was prepared using this purified IgMmonoclonal antibody following the procedures described by Pharmacia FineChemicals. Sufficient (2.0 mg) monoclonal antibody was dissolved incoupling buffer comprising 0.1M NaHCO₃, pH 8.3 and 0.5M NaCl to form a1.0 mg/ml solution. This antibody solution was added to 2.5 g ofcyanogen bromide activated Sepharose-4B (Sigma) which had been incubatedpreviously in 8.0 ml of 0.001M HCl overnight. After the Sepharose andantibody solution were allowed to react for 1 hour, the unbound sites ofthe antibody bound gel were blocked by incubating the solid phasesorbant material with 1.0M ethanolamine, pH 8.5 for 1 hour. Thecombination of the IgM antibody immobilized onto the solid phase sorbantmaterial formed an affinity matrix which then was used in volumes offrom 1-2 ml.

Although the preferred solid phase sorbant material is the activatedSepharose 4B gel, it is recognized that many other materials may besubstituted as the solid phase material. These include other agarose gelcompositions, dextrans, carbon and silicon granular preparations and thelike, including glass plates. Similarly, methods for immobilizing thehigh affinity IgM antibody onto each of these different chemicalcompositions are known and described in the art. For this reason, thepreparation of a solid phase sorbant material which is then coupled tothe high affinity IgM antibody as described herein in any form,concentration, or configuration is deemed to be within the scope of thepresent invention.

V. Methodology for Detecting and Isolating Aflatoxins in a Fluid Sample

The methodology for detecting aflatoxins and aflatoxin-DNA adducts in atest sample is an affinity chromatography technique comprising thefollowing steps: Preparing an affinity matrix comprising a homogenous,high affinity IgM antibody specific for the aflatoxin or aflatoxin-DNAadduct of interest which has been immobilized onto a solid phase sorbentmaterial; combining the test sample with the affinity matrix such thatthose aflatoxins as are present in the test sample become bound to andare retained by the IgM antibodies of the affinity matrix; adding areleasing agent to the affinity matrix for the release of the aflatoxinfrom the IgM antibodies, this releasing agent comprising at least a 50%(v/v) aqueous solution of a compound selected from the group consistingof dimethyl sulfoxide, dimethyl formamide and dimethyl acetamide; andidentifying the presence of the aflatoxin in the effluent collected fromthe affinity matrix.

A critical characteristic of the anti-aflatoxin IgM monoclonalantibodies is not only its specificity for this AFB₁ antigen but alsoits high affinity constant (at least about 1×10⁸, and preferably atleast about 1×10⁹, liters per mole) which requires the use of uniquereleasing agents for the detachment of the aflatoxin of interest afterit has become bound to the covalently linked antibody in the affinitycolumn. This is demonstrated by the following examples which identifynot only the ability of the antibody bound sorbant material to bind theaflatoxin of interest but also to indicate the inability of releasingagents and eluants known in the art to selectively detach and releasethe aflatoxin after it has become bound within the affinity column.

EXAMPLE 1

Initial experiments were performed to determine the ability of anti-AFB₁IgM antibody immobilized upon Sepharose 4B as an affinity material tobind aflatoxin B₁. In these studies radio-labelled ³ H-aflatoxin B₁ wasused at a concentration of 1 nanogram in 10 ml PBS aliquots, a levelwhich would approximate the concentration of aflatoxins expected to befound in the body fluids of humans exposed to contaminated foods.Initially 100 μl of ³ H-AFB₁ tracer in 10.0 ml containing 0.1% BSA, 10mM NaN₃, 0.1 ml normal mouse sera and 3 μl of ³ H-AFB₁ was added toabout a 1.0 ml gel bed. The eluant column flow rate was slightly greaterthan 0.25 ml per minute. About 200 μl of PBS was added to the tracer andtransferred to the column by allowing it to run down the column untilthe top of the affinity matrix was exposed to the air (without allowingthe column to dry out). An additional 2.0 ml of PBS was added to washthe tracer through the column and the effluent was collected in a vialas a single fraction (No. 1). In this way approximately 24,486 CPM oftracer material was added to the column and, as each fraction wascollected, the effluent analyzed for radio-labelled content byscintillation. Different eluants were then added to the affinity matrixto determine their ability to release the AFB.sub. 1 tracer. The resultsare given in Table 1 below.

                  TABLE I                                                         ______________________________________                                                                            CPM                                       Effluent               Volume       Per                                       Fraction                                                                              Eluant         Used         Fraction                                  ______________________________________                                                Sample & PBS   300 ul + 2.0 ml                                                                            2,494                                     2       PBS Wash       2.0 ml       287                                       3       2M KSCN        3.0 ml       347                                       4       PO.sub.4 buffer/2.64 M                                                                       3.0 ml       122                                               NaCl, pH 3.0                                                          5       DEA buffers/   3.0 ml       36                                                2.64M NaCl,                                                                   pH 9.9                                                                6       PO.sub.4 buffer/                                                                             2.0 ml       38                                                0.14M NaCl,                                                                   pH 2.0                                                                7       50% DMSO/PO.sub.4                                                                            2.0 ml       23,632                                            Buffer/0.14 NaCl                                                              pH 2.0                                                                ______________________________________                                         Total recovery of radioactivity was 100% within experimental error.      

As is evident, about 10% quantitatively of the original tracer was notadsorbed by the affinity matrix initially and was collected in effluentfraction No. 1 prior to the addition of any of the eluants tested. Thesefindings have been attributed to non-specific tritium exchange in thetracer. Subsequent reaction with each eluant listed revealed nosubstantial release of tracer AFB₁ by the IgM antibodies of the matrixin effluent fractions 2-6. It was only after using 50% dimethylsulfoxide (hereinafter "DMSO") that the bulk (90%) of the AFB₁ tracerwas released from the affinity matrix. It is also noted that when theDMSO was added to the affinity column, a definite color change withinthe matrix became visible and the column bed itself became moretranslucent. When PBS was added to the affinity matrix after the DMSOeffluent fraction was collected, the gel bed returned to its originalopaque state and normal condition.

It will be appreciated also that the affinity matrix material returnedto its original normal condition without damage to or modification of(by denaturation, precipitation, etc.) the IgM antibodies conjugated tothe Sepharose gel in any way. This is demonstrated by the continuationof this experiment after the DMSO releasing agent had beenadded-followed by a washing with PBS.

A second 100 μl quantity of tracer ³ H-AFB₁ dissolved in 200 μl of PBSwas then added to the same column which had been previously used toisolate the first sample of ³ H-AFB₁ tracer. The second tracer aliquotwas the followed by a rinse of 2.0 ml PBS and the effluent collected asfraction No. 8. A 2.0 ml aliquot of DMSO in PBS was then added and theeffluent collected as fraction No. 9. The column was then washed withtwo additional 2.0 ml PBS aliquots and the effluents collected asfractions 10 and 11. The results are given in Table II below.

                  TABLE II                                                        ______________________________________                                        Effluent                                                                      Fraction                        % of                                          No.        Eluant       cpm     Original                                      ______________________________________                                        8          PBS          2,658   ˜10%                                    9          50% DMSO     14,462  ˜60%                                    10         1st PBS wash 8,282   ˜30%                                    11         2nd PBS wash 530     ˜0%                                     ______________________________________                                    

The second tracer aliquot contained approximately 24,486 cpm as thetotal radio-label load and it will be noted that approximately 10% ofthe tracer was not retained by the affinity matrix again. Fraction 9again indicates that the bulk of the AFB₁ tracer was released by theDMSO (about 60%) while the remainder was collected in fraction 10 as thefirst subsequent PBS washing containing some residual DMSO.

In addition, a variation of this experiment was conducted to show theneed for using at least a 50% concentration of DMSO before the releasingeffect can be obtained. For this experiment, the 100 μl tracer aliquotof ³ H-AFB₁ contained approximately 27,000-28,000 cpm in total. The 100μl aliquot of tracer was loaded on to the affinity column followed by aninitial washing of 200 μl of PBS to remove the non-retained tracer fromthe column. The matrix was then washed with increasing concentrations ofDMSO in 1.0 ml aliquots. The results are given in Table III below.

                  TABLE III                                                       ______________________________________                                        Effluent                                                                      Fraction              Volume      Effluent                                    No.        Eluant     Used        cmp                                         ______________________________________                                        1          PBS        1.0 ml      455                                         2          PBS        1.0 ml      2,271                                       3          1% DMSO    1.0 ml      189                                         4          1% DMSO    1.0 ml      79                                          5          5% DMSO    1.0 ml      45                                          6          5% DMSO    1.0 ml      82                                          7          10% DMSO   1.0 ml      117                                         8          10% DMSO   1.0 ml      92                                          9          20% DMSO   1.0 ml      41                                          10         20% DMSO   1.0 ml      56                                          11         30% DMSO   1.0 ml      86                                          12         30% DMSO   1.0 ml      95                                          13         40% DMSO   1.0 ml      72                                          14         40% DMSO   1.0 ml      50                                          15         50% DMSO   1.0 ml      88                                          16         50% DMSO   1.0 ml      15,682                                      17         PBS        1.0 ml      5,966                                       18         PBS        1.0 ml      49                                          ______________________________________                                    

It is apparent by the results in Table III that there is no substantialrelease of ³ H-AFB₁ from the affinity matrix prior to using a 50%solution of DMSO. It was found also that concentrations of DMSO greaterthan 50% did not measurably increase the rate or quantity of AFB₁released from the affinity matrix. For this reason, any concentration ofDMSO of at least 50% is sufficient for use as the releasing agent inthis methodology. Other releasing agents found suitable for use in thismethodology were dimethyl formamide and dimethyl acetamide; each ofthese should be used in a concentration of at least 50% in order to beeffective as a releasing agent. The use of these releasing agents, oraprotic solvents in general, permits the affinity matrix or column to bereusable for numerous sample runs. Eluting with such aprotic solventsdoes not cause deterioration of the matrix material, which may be ofimportance to industrial applications for the detection and measurementof mycotoxins. Economical use of the process in industrial applicationswould require a reusable affinity matrix and elution with aproticsolvents, such as DMSO, meets this need.

On the other hand, elution can also be performed with lipophilicsolvents, such as methanol and ethanol, in which case the affinitymatrix or column will be substantially nonreusable. According to thisaspect of the invention, elution is conducted with preferably 50-100%methanol or ethanol, particularly 100%, proceeding similar to theprocedure described above, except substituting methanol or ethanol forDMSO.

VI. In Vitro Isolation of Aflatoxin from Human Urine, Serum and Milk

As demonstrated above, the capacity of the IgM antibody affinity matrixto bind AFB₁, as determined by radiometric and absorbance technique, wasto bind 1.0-1.3 μg of AFB₁ from 10 ml of PBS per 1 ml of column bedvolume. These parameters may be used to isolate and detect aflatoxins invitro from human urine, serum and milk samples as follows:

Human serum (10 ml aliquots) or human milk (10 ml aliquots) may beapplied directly to the antibody matrix without prior treatment of thesample. In both instances, quantitative binding of aflatoxins in thefluid sample to the antibody matrix occurs which are then quantitativelyrecovered using a 50% concentration of DMSO as the releasing agent. Thishas been empirically determined using ³ H-AFB₁ in 1 nanogramconcentrations added directly to the serum or milk samples in the mannerearlier described. Therefore, when testing either serum or milk testsamples, no preparative steps are required for quantitative recovery ofthe aflatoxin.

Urine samples however require a preparative step involving preparativechromatography to remove interfering materials, e.g. salts, within thesample before isolation and recovery of the aflatoxin can be achieved.This is shown schematically in FIG. 3 in which a Sep-Pak C₁₈ cartridge(Waters Associates, Inc.) is preferably used to remove the interferingmaterials. If the urine sample is first passed through a preparative,low pressure, liquid chromatography cartridge to remove proteins andother interfering materials, 90-95% of the aflatoxins present in humanurine samples are consistently and quantitatively recovered using thepresent methodology.

It should be noted also that the use of high affinity IgM antibodyspecifically raised against AFB₁ is also useful for the detection andrecovery of the AFB₁ -DNA adduct, AFB₁ -N⁷ -Gua on a quantitative basis.These aflatoxin DNA adduct products can be detected without anypreparative step and recovered quantitatively from serum and otherbodily fluids. If urine from humans and animals comprise the test sampleit is preferred that the preparative step of HPLC through a carbonchromatography cartridge be performed initially before adding the sampleto the affinity matrix. In this manner, a quantitative and qualitativemethod is available to isolate and purify aflatoxin B₁ and itsmetabolites from complex biological samples obtained fromenvironmentally exposed populations. In addition, it also serves as ananalytical methodology for the detection of the aflatoxin itself.

From the above, it can be seen that the present invention provides ameans for easily and accurately detecting levels of a toxin,particularly aflatoxin, in a sample. An important aspect of theinvention is the affinity matrix or column to which is bound thespecific monoclonal antibody. The use of such an affinity matrixcomprised of antibody immobilized onto a solid phase sorbant provides ameans for quick, efficient high purification of samples for testing.Because of the high affinity and selectivity characteristics of themonoclonal antibody, a crude ground extract of a sample can be runthrough the antibody-matrix column, whereby the toxin of interest willbind to the antibody while all remaining ingredients of the sampleextract will pass through. Subsequent elution with an appropriatereleasing agent, such as DMSO or methanol, will result in an eluting ofthe toxin into a collected sample which will contain highly purifiedtoxin in quantitative yields from the original crude sample. Such anefficient one-step purification procedure is an important advance,particularly in the field of assays for toxins.

This important aspect of the invention has broad applications to thedetection and measurement of other toxins, particularly mycotoxins, bythe use of a matrix or column having bound thereto a monoclonal antibodyspecific for the toxin of interest. Once the toxin has been purified bymeans of the antibody-matrix material, detection of the toxin can bemade, as desired, by various qualitative or quantitative assaymeasurement techniques, such as fluorescence, immunoassay,radioimmunoassay, ELISA method, TLC, HPLC, gas chromatography,colorimetric analysis, and the like. Selection of the particulardetection method will depend upon a variety of factors, including typeof toxin, expected level of toxin and the use for the detected data. Ineither event, the present invention provides a method utilizing anaffinity matrix having bound thereto a monoclonal antibody wherebysamples for analysis and detection can be prepared simply and quickly bya single pass through the affinity matrix.

For aflatoxins, one preferred detection method is fluorescence, wherebythe purified sample is subjected to UV light (365 nm) and the aflatoxinfluoresces at 425 nm. This type of analysis is particularly preferredbecause of its easy performance and yet its usefulness in even providingsome level of quantitative detection. These fluoresence analysisprocedures can be conducting following AOAC methodology (see AOACMethods, Ch. 26, pp. 414-429 (1980) hereby incorporated by reference).

Because the use of the antibody bound matrix permits such improvedone-step purification of a sample containing toxin, the invention isapplicable to a variety of samples which could be tested for levels oftoxins, including industrial wastes and foods, such as corn, peanuts andthe like, as well as body fluids. A general flow diagram for the overallprocess is set forth below, exemplified by use of a corn sample.##STR1##

From this flow diagram, it is clear that the important common stepresides in purification of the sample by use of the affinity columnhaving the specific monoclonal antibody bound thereto. Once the sampleis purified, the toxin level or presence can be qualitatively orquantitatively measured by a variety of means, and all or part or noneof the procedure can be automated.

Qualitative Assay

Toxin levels according to the invention can first of all be measuredqualitatively. As a particular example, aflatoxin levels in a cornsample can be detected according to the following process steps:

1. Weigh-out 50 gms of ground corn

2. Place ground corn in blender

3. Add Methanol to blender

4. Blend for 1 minute

5. Add to blended mix Distilled Water and mix

6. Draw extract from blender through a filter

7. Deliver extract through aflatoxin affinity column

8. Deliver Distilled Water through aflatoxin affinity column

9. Deliver Methanol through aflatoxin affinity column (hopefullycontained in a syringe) into reaction vessel containing means forfluorescent illumination

10. Remove vessel containing means for fluorescent illumination andplace in apparatus for visually determining fluorescence level (notquantitative)

11. Remove and dispose-of vessel

Quantitative Assay

On the other hand, toxin level can be quantitatively measured accordingto the following procedure, again as exemplified with aflatoxinmeasurement in corn:

1. Weigh-out 50 gms of ground corn

2. Place ground corn in blender

3. Add Methanol to blender

4. Blend for 1 minute

5. Add to blended mix Distilled Water and mix

6. Draw extract from blender through a filter

7. Deliver extract through aflatoxin affinity column

8. Deliver Distilled Water through aflatoxin affinity column

9. Deliver Methanol through aflatoxin affinity column into test cuvettecontained in measuring apparatus

10. Read results

11. Automated disposal of liquid sample

The above qualitative and the quantitative procedures can be performedmanually, partially automated or fully automated. FIG. 4 shows anautomated system for aflatoxin assays, comprising essentially a blendingmeans 2, a filter means 4, an affinity column 6, an eluent metering anddispensing means and a detection means 8 for either qualitative orquantitative detection of aflatoxin.

The invention is not to be restricted in form nor limited in scopeexcept by the claims appended hereto.

What we claim is:
 1. A method for detecting toxins in a test sample,said method comprising:preparing a sample believed to contain a toxin;placing said sample on an affinity column comprised of a solid phasesorbant material and, immobilized thereon, a monoclonal antibodyspecific for said toxin; eluting said column with a solvent to recover afirst eluent, whereby said toxin is retained on said column by saidmonoclonal antibody and separated from all other ingredients of saidsample; eluting said column with a releasing agent to recover a secondeluent, whereby said toxin is released from said antibody and recoveredin said second eluent; and subjecting said second eluent to fluorescencemeasurement by exposing said second eluent to UV light for detection ofthe presence of said toxin, with the proviso that said second eluent isnot subjected to high pressure liquid chromatography or otherpurification subsequent to recovery from said column.
 2. A methodaccording to claim 1, wherein said releasing agent comprises a not lessthan 50% solution of an aprotic solvent.
 3. A method according to claim2, wherein said aprotic solvent is dimethyl sulfoxide, dimethylformamide or dimethyl acetamide.
 4. A method according to claim 1,wherein said releasing agent comprises a 50-100% solution of alipophilic solvent.
 5. A method according to claim 4, wherein saidsolvent comprises 100% methanol or 100% ethanol.
 6. A method accordingto claim 5, wherein said toxin is aflatoxin B₁ or aflatoxin M₁ and saidantibody is specific for said aflatoxins with an affinity of at leastabout 1×10⁸ liters per mole.
 7. A method according to claim 6, whereinsaid second eluent is exposed to UV light at 365 nm for visual detectionof the presence of aflatoxin.
 8. A method according to claim 6, whereinsaid second eluent is subjected to quantitative analysis for determiningthe level of aflatoxin in said sample.
 9. A method according to claim 1,wherein said toxin is a mycotoxin having a molecular weight of not morethan about 1,000 daltons.
 10. A method according to claim 9, whereinsaid toxin is a member selected from the group consisting of aflatoxins,fluoranthene, nitropyrene, nitrosopyrene, nitrofluoranthene,nitrochrysene, aminobiphenyl, and their respective conjugates andderivatives.
 11. A method according to claim 1, wherein said sample isserum, urine or food product.
 12. A method according to claim 11,wherein said food product is milk, corn or peanut products.
 13. A methodfor detecting aflatoxins in a test sample, said methodcomprising:placing a sample believed to contain aflatoxin on an affinitycolumn comprised of a solid phase sorbant material and, immobilizedthereon, a monoclonal antibody for said aflatoxin; eluting said columnwith a solvent to recover a first eluent, whereby said aflatoxin isretained on said column by said monoclonal antibody and separated fromall other ingredients of said sample; eluting said column with areleasing agent to recover a second eluent, whereby said aflatoxin isreleased from said monoclonal antibody and recovered in said secondeluent; and subjecting said second eluent to fluorescence measurement byexposing said second eluent to UV light for detection of the presence ofsaid aflatoxin, with the proviso that said second eluent is notsubjected to high pressure liquid chromatography or other purificationsubsequent to recovery from said column.
 14. A method according to claim13, wherein said releasing agent comprises a not less than 50% solutionof an aprotic solvent.
 15. A method according to claim 14, wherein saidaprotic solvent is dimethyl sulfoxide, dimethyl formamide or dimethylacetamide.
 16. A method according to claim 13, wherein said releasingagent comprises a 50-100% solution of a lipophilic solvent.
 17. A methodaccording to claim 16, wherein said solvent comprises 100% methanol or100% ethanol.
 18. A method according to claim 13, wherein said antibodyhas an affinity of at least about 1×10⁸ liter per mole for the aflatoxinof interest.
 19. A method according to claim 13, wherein said samplecomprises serum, urine or food product.
 20. A method according to claim19, wherein said food product is milk, corn or peanut products.
 21. Amethod according to claim 13, wherein said second eluent is exposed toUV light at 365 nm.
 22. A method according to claim 13, wherein saidsecond eluent is subjected to quantitative analysis for determining thelevel of aflatoxin in said sample.
 23. A method for detecting aflatoxinsin a test sample, said method comprising:placing a sample believed tocontain aflatoxin on an affinity column comprised of a solid phasesorbant material and, immobilized thereon, a monoclonal antibody forsaid aflatoxin; eluting said column with a solvent to recover a firsteluent, whereby said aflatoxins are retained on said column by saidantibody and separated from all other ingredients of said sample;eluting said column with a releasing agent comprised of ethanol ormethanol to recover a second eluent, whereby said aflatoxins arereleased from said antibody and recovered in said second eluent; andsubjecting said second eluent to fluorescence measurement by exposingsaid second eluent to UV light at 365 nm for detection of the presenceof aflatoxins, with the proviso that said second eluent is not subjectedto high pressure liquid chromatography or other purification subsequentto recovery form said column.
 24. A method according to claim 23,wherein said antibody has an affinity of at least about 1×10⁸ liters permole to said aflatoxins.
 25. A method according to claim 23, whereinsaid sample comprises serum, urine or food product.
 26. A methodaccording to claim 25, wherein said food product is milk, corn or peanutproducts.